The three-dimensional structure of proteins determines their biological activities. Protein misfolding may result in the production of defective proteins, that may lead to dramatic symptoms as those observed in genetic diseases and certain types of cancers. GRP78/BiP is a molecular chaperone, which belongs to the highly conserved family of 70 kDa heat shock proteins (HSP70) and plays a role in the folding and assembly of secreted and membrane proteins. The physiological functions of HSP70s are believed to be the result of their ability to bind to unfolded polypeptides, to hydrolyze ATP, and to interact with accessory proteins that regulate their enzymatic activity and affinity for peptidic and nucleotidic substrates. Our goal is to investigate the interplay of ATP hydrolysis, recognition of unfolded polypeptides, and interactions with accessory proteins in the chaperone function of BiP, by using a combination of biochemical, spectrometric, structural and mutational approaches. Our preliminary data indicate that the ATPase activity and binding specific unfolded peptidic sequences are carried out by the N- and C- terminal domains, respectively. We have characterized the C-terminal domain of GRP78/BiP and found that its specificity towards peptides can be significantly altered by site-directed mutagenesis. In order to gain more information about the role of then peptide binding domain of BiP, we will determine if the peptide specificity of the C- terminal domain determines BiP function in protein folding. Mutants that exhibit altered equilibrium or kinetic constants for the binding of unfolded model peptides will be assayed for their ability to promote refolding of chemically denatured polypeptides. The determination of the structure of the peptide binding domain in solution will be determined by using NMR techniques, in a collaborative project, and dynamic studies will be performed to characterize, at the molecular level, the interactions between BiP and model synthetic peptides. To understand how the ATPase and chaperone activities of BiP relate to each other we will study biochemically at which step(s) of the BiP regulatory cycle the N-and C-terminal domains interact with each other. We will also map the interactions of GRP78/BiP with accessory proteins and analyze whether accessory proteins stimulate the ATPase activity of the N-terminal domain, and /or modulate the affinity of the C-terminal domain for peptidic substrates. Finally, we will investigate whether the peptide binding domain of GRP78/BiP can inhibit or compete with some of the catalytic or biological functions of the E. coli HSP70 homologue, DnaK. This investigation will characterize the role of specificity for unfolded peptidic substrates, affinity for adenine nucleotides and for accessory proteins in the functions of GRP78/BiP in assisted protein folding.